Our goal is to determine the structural basis and mechanism of retroviral genome selection and packaging. During the current funding period we (1) showed that genome packaging by the Moloney Murine Leukemia Virus (MoMuLV) is mediated by a dimerization-dependent structural switch, (2) determined the 3D structure of a MoMuLV core encapsidation element (132 nucleotide dimer) using a novel NMR/cryo-electron tomography (cryo-ET) approach, (3) developed a 2H- and 13C-edited NMR method that enabled direct NMR detection of structural elements within the intact HIV-1 5'-leader (712 nucleotide dimer); (4) demonstrated that HIV-1 genome dimerization and packaging are regulated by a novel allosteric nucleotide displacement RNA switch mechanism, in which base pairing of residues near the gag start codon with those of an upstream element induces a global RNA rearrangement that simultaneously exposes a dimer-promoting stem loop and high affinity nucleocapsid (NC) binding sites; and (5) identified a minimal HIV-1 core encapsidation signal that exhibits NMR, dimerization, and NC binding properties of the intact leader and can efficiently direct the packaging of heterologous cytoplasmic RNAs into assembling particles. In addition, we have obtained unpublished NMR evidence that the HIV-1 packaging signal adopts a novel 3D structure that is unlike any of the > 25 structures predicted previously on the basis of chemical reactivity probing and modeling. We are now on the verge of completing the 3D structure of the HIV-1 core encapsidation signal which, when finished, will provide insights into the RNA structures and protein-RNA interactions that direct HIV-1 genome selection. We have also developed NMR tools that enable identification of intermolecular contacts in the native, dimeric HIV- 1 5'-leader, and we are poised to determine the 3D structure of the full-length HIV-1 5'-leader in its monomeric and dimeric states. Preliminary ITC and biophysical studies indicate that an HIV-1 Gag fragment comprising the CA-through-NC domains (GagCANC) binds the minimal packaging signal RNA with high affinity and 12:2 Gag:RNA stoichiometry. By using our hybrid NMR/cryo-ET approach, it should now be possible to determine the structure of the Gag:RNA complex that is trafficked to the plasma membrane and nucleates virus assembly. NMR studies of the monomeric HIV-1 5'-leader and a spliced HIV-1 mRNA will provide insights into the interactions and mechanisms that block packaging of these RNAs, and comparisons with HIV-2 and MoMuLV will help establish if RNA-centered control mechanisms are evolutionarily conserved. NMR studies of large RNAs are technically challenging - the average size of NMR-derived RNA structures in the RNA Structure Database is only 27 nucleotides - but the potential payoff is substantial and could ultimately lead not only to a more detailed understanding of how HIV replicates, but also to the development of new approaches for the treatment of AIDS, cancers, and other virally-induced human diseases.